Typically, proteins are purified using conventional chromatography or high-performance liquid chromatography methods. Chromatographic methods that can be used to purify proteins include gel-filtration chromatography, ion-exchange chromatography, immunoaffinity chromatography and metal-chelate affinity chromatography. The high-performance liquid chromatographic methods that can be used to purify proteins include reverse-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, size-exclusion high-performance liquid chromatography and high-performance chromatofocusing and hydrophobic interaction chromatography.
Purification of proteins by conventional chromatography is usually achieved using a combination of chromatographic methods including gel-filtration, ion-exchange, hydrophobic-interaction, dye-interaction, affinity and immunoaffinity chromatography. With the possible exception of affinity or immunoaffinity chromatography, it is rarely possible to purify a protein to homogeneity in a single chromatographic step (see Current Protocols in Molecular Biology, Vol. 2, 10.9.2, Ausubel et al., eds. 1993). If affinity or immunoaffinity columns are not available for purifying a particular protein, then it is typically necessary to utilize sequential chromatographic steps and to analyze (such as by one-dimensional gel electrophoresis) the protein after each purification step, to determine if the protein is homogeneous. Id.
Purification of proteins by high-performance liquid chromatography (HPLC) relies on the use of rigid, small-particle matrices at high operation pressure. Id. at 10.12.1. Unlike conventional chromatography, HPLC is most suitable for purifying low-microgram quantities of proteins collected in small fraction volumes with a short separation time. Id. The limitation of small sample load results from the low-loading capacity of commercially available, analytical HPLC columns. Id. Typically, HPLC is used most frequently in later stages of protein purification, typically after one or more conventional chromatographic separations have been used to reduce the mass of contaminating proteins and to simplify a complex protein mixture. Id.
As discussed earlier, one of the problems with conventional chromatography or high-performance liquid chromatography methods is that it is rarely possible to purify a protein to homogeneity in a single step using either of these methods. Additionally, conventional chromatography or high performance liquid chromatography methods are labor intensive and these methods are expensive to use.
Thereupon, there is a need in the art for a process that can be used to purify proteins to homogeneity in a single step, is inexpensive to use and is not labor intensive.